Acyl Carrier Proteins (ACPs) play important roles in a number of biosynthetic pathways that are dependent upon acyl group transfers. They are most often associated with the biosynthesis of fatty acids [1,2], but they are also utilized in the synthesis of polyketide antibiotics [3,4], non-ribosomal peptides [5,6], and of intermediates used in the synthesis of vitamins such as the protein-bound coenzymes, lipoic acid [7] and biotin [8]. The ACP in each of these pathways is composed of 80-100 residues and is either an integrated domain in a larger multi-functional protein (Type I) or is a structurally independent protein that is part of a non-aggregated multi-enzyme system (Type II). Type I ACPs are found in mammals, fungi and certain Mycobacteria while type II ACPs are utilized by plants and most bacteria. The fact that these proteins are essential for the maturation of the organism has led to their investigation as targets for the development of new anti-microbial agents [9-12].
ACPs require post-translational modification for activity. They are converted from an inactive apo-form to an active holo-form by the transfer of the 4′-phosphopantetheinyl (P-pant) moiety of coenzyme A to a conserved serine on the ACP. Evidence now suggests [13] that each ACP that is dependent upon P-pant attachment for activation has its own acyl carrier protein synthase responsible for this attachment.
The post-translational modification of the fatty acid synthase ACP is performed by holo-[acyl carrier protein] synthase (hereinafter defined as “ACPS”; Enzyme Commission No. 2.7.8.7). ACPS produces holo-fatty acid synthase ACP by transferring the P-pant moiety to Ser-36 of the apo-fatty acid synthase ACP in a magnesium dependent reaction [14] as follows: 
The over-expression and purification of the ACPS from Escherichia coli has been described [15] and this protein has been classified as a member of a new enzyme superfamily, the phosphopantetheinyl transferases [13]. Other members of this superfamily have low similarity with E. coli ACPS (12-22%), but each has been shown to possess P-pant transferase activity. Alignment of these proteins show that two regions, residues 5-13 and 51-65 (E. coli ACPS numbering), are highly conserved with eight of the residues in these regions being strictly conserved.
While numerous members of the phosphopantetheinyl transferase superfamily have been identified and sequenced, until the present invention, no one, to the inventors' knowledge, has discovered the crystal structure of an ACPS-like phosphopantetheinyl transferase or has characterized the three dimensional structure of the molecule's Co-A active site. Determination of the three dimensional structure of ACPS and its CoA active site is critical to the rational identification and/or design of therapeutic or antibiotic agents that may act as inhibitors or activators of ACPS enzymatic activity. Alternatively, using conventional drug assay techniques, the only way to identify such an agent is to screen thousands of test compounds, either in culture or by administration to suitable animal models in a laboratory setting, until an agent having the desired inhibitory or activating effect on a target compound is identified. Necessarily, such conventional screening methods are expensive, time consuming, and do not elucidate the method of action of the identified agent on the target compound.
However, advancing X-ray, spectroscopic and computer modeling technologies allow researchers to visualize the three dimensional structure of a targeted compound. Using such a three dimensional structure, researchers identify putative binding sites and then identify or design agents to interact with these binding sites. These agents are then screened for an activating or inhibitory effect upon the target molecule. In this manner, not only are the number of agents to be screened for the desired activity greatly reduced, but the mechanism of action on the target compound is better understood. Further, the three dimensional structure of one compound determined using these techniques can be used to ascertain the three dimensional structure of other related compounds.
Recently, Reuter, et al. have disclosed the crystal structure of another member of the P-pant transferase superfamily, Sfp, complexed with CoA [22]. Sfp converts the inactive apo forms of the seven PCP domains of surfactin synthetase to their active holo-forms by transfer of the 4′-phosphopantetheinyl moiety of CoA to the side chain hydroxyl of a serine residue found in PCP domains. Thus, Sfp is essential in the production of lipoheptapeptide antibiotic surfactin in B. subtilis [22].
The “Sfp-like” P-pant transferases are very different than the “ACPS-like” P-pant transferases. In particular, the Sfp-like P-pant transferases activate PCP (peptidyl carrier protein) domains of various non-ribosomal peptide synthetases and are present in monomeric form. Further, Sfp shows a pseudo 2-fold symmetry dividing the molecule into two similarly folded halves of roughly identical size. In contrast, the ACPS-like P-pant transferases, which form homodimers, activate the ACP domains or subunits of fatty acid synthetases, polyketide synthases and other enzymes, and are about half the size of Sfp-like transferases. While the pseudo 2-fold symmetry of the Sfp synthetase activating enzyme disclosed by Reuter, et al. suggests that dimerization may be necessary for the formation of an intact ACPS-like P-pant transferase [22], the crystal structure of Sfp, alone or complexed with CoA, is not sufficient to generate a three dimensional model of an ACPS-like P-pant transferase nor is it useful for designing or identifying agents which may activate or inhibit ACPS enzymatic activity. Furthermore, as discussed below, there are significant differences between the Sfp and ACPS structures that clearly place the two enzymes in different functional groups of the P-pant transferase superfamily.